The experiments outlined in this proposal are focused at studying the mechanism of thyrotropin releasing hormone (TRH) biosynthesis in primary cultures of hypothalamic neurons. The Specific Aims of the proposed research are to: 1) establish serum-free culture conditions for the maintenance of primary hypothalamic neurons that express high levels of TRH 2) utilize antisera specific for TRH-Gly and TRH and biosynthetic labeling to demonstrate that the glycine-extended peptide functions as the direct precursor to the mature amidated peptide. 3) begin studies that extend the above aims to identify and characterize intermediates of the TRH biosynthetic pathway that are larger than TRH-Gly The general approach will involve demonstrating the efficient incorporation of labeled amino acids into TRH by cultured cells only when the media are supplemented with ascorbic acid, the proposed co-factor for Alpha-amidation. In the absence of the co-factor the proposed alternate peptide product is TRH-Gly. Techniques involving the biosynthetic labeling of proteins in cell cultures will be coupled with several types of HPLC (seze exclusion, reversed phase, ion exchange), several methods of peptide mapping (pyroglutamate aminopeptidase, and peptidylglycine Alpha-amidating monoxygenase), and immunopurification of labeled peptides with antibodies specific for TRH or TRH-Gly. Pulse-chase experiments will be used to test whether TRH-Gly is converted to TRH in an ascorbic acid-dependent manner. The long-term goal of these studies is to describe the roles of neuropeptides in CNS function and thus to establish the importance of neuropeptides in mental health.